Jun 20, 2025
Assay for lysosomal activity using Magic Red
- Bishal Basak1,2,
- Erika Holzbaur1,2
- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
- University of Pennsylvania
Protocol Citation: Bishal Basak, Erika Holzbaur 2025. Assay for lysosomal activity using Magic Red . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8q437l2w/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 20, 2025
Last Modified: June 20, 2025
Protocol Integer ID: 220604
Keywords: lysosomal activity, using magic red, assay, magic red
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
Assay for lysosomal Cathepsin B activity in neurons using Magic Red
Protocol materials
Magic Red substrateBio-Rad LaboratoriesCatalog #ICT937
Plate neurons
Plate 120,000-150,000 neurons in an imaging dish at days in vitro (DIV) 0
Note
Protocol for primary neuron isolation and in vitro culture can be obtained from:
(DIV) 0
Preparing reagents
Reconstitute Magic Red substrate upon arrival in 50 µL of DMSO Magic Red substrateBio-Rad LaboratoriesCatalog #ICT937
On the day of imaging prepare 1:10 fresh dilution of reconstituted Magic Red solution in water
On the day of imaging equilibrate 4 ml of fresh maintenance media (MM) and 4 ml of imaging media (IM) per imaging dish in an incubator
Note
Maintenance media composition can be obtained from: dx.doi.org/10.17504/protocols.io.81wgby723vpk/v1
Imaging media composition can be obtained from:
Imaging steps
15m
At DIV 6 or 7, aspirate out conditioned media from the imaging dish, wash the neurons once with fresh MM
Add 2 mL of fresh MM and then add 5 µL of the diluted Magic Red solution (prepared in step 3)
Incubate the neurons in an incubator at 37 °C for 00:15:00
15m
Post incubation, aspirate out the media, and wash once with fresh IM
Add 2 mL of fresh IM and then add 5 µL of the diluted Magic Red solution (prepared in step 3)
Image neurons live under a confocal microscope with a heating chamber kept at 37 °C